Micro-organisms produce a wide variety of compounds which demonstrate anti-bacterial properties; one group of these compounds, the bacteriocins, consists of bactericidal proteins with a mechanism of action similar to the ionophore antibiotics. Bacteriocins are often active against species which are closely related to the producer. Their widespread occurrence in bacterial species isolated from complex microbial communities (for example, the intestinal tract, the oral or other epithelial surfaces) suggests that bacteriocins may have a regulatory role in terms of population dynamics within bacterial ecosystems.
Bacteriocin production is widespread throughout the Eubacteria, particularly in species occupying niches in complex microbial communities. Bacteriocin production may be important in determining dominance, colonisation and maintenance of continuity in such communities. Although scant information is available concerning the occurrence of bacteriocins in rumen bacteria, it is likely that they will be of considerable importance. The rumen may also be a source of interesting new bacteriocins, with unusual spectra of activity. As such, a survey for ruminal bacteriocin producers and an evaluation of the effects of bacteriocins on the endogenous microbial population may provide useful information in our overall understanding of rumen ecology as well as allowing the identification of new antimicrobial compounds for use in a wide range of applications.
Ruminal bacteriocins may be particularly suitable for applications in ruminant production systems. A wide variety of compounds useful for the manipulation of the microbial ecology of the rumen have been investigated. Among the most effective for gross alteration of the rumen fermentation profile are the ionophore antibiotics. These antibiotics work by selecting against the Gram Positive rumen component. Reduction in this rumen component results in an alteration of the acetate to propionate ratio of fermentation end-products, reduction in methane generation, and a significant increase in the efficiency of the rumen fermentation, as reflected by decreased feed inputs. Furthermore, these agents have application for alteration of both dairy milk fat quantity and quality.
The ratio of acetate to propionate produced in the rumen appears to be the primary determinant of butterfat levels in milk. This ratio is determined by the relative numbers of acetate-producing bacteria and propionate-producing bacteria in the rumen. The ionophore antibiotics used to manipulate the ratio of acetate to propionate producing bacteria in the rumen are produced as fermentation products by micro-organisms (streptomycetes) originally isolated from soils. Ionophore antibiotics have a history of effective use in meat production systems, but are not presently used in dairy production. Ionophore antibiotics have a number of limitations when used in ruminant production systems. These disadvantages include that:
(a) they are not produced by the rumen bacteria themselves and must be introduced into the rumen repeatedly, PA1 (b) they are not proteins and therefore they are not subject to easy modification by genetic engineering and are not easily digested and utilised by the animal like a protein (in fact they may exhibit residue problems), and PA1 (c) they do not exhibit a high degree of specificity for their targets and thus they do not provide opportunities for precise control of rumen output.
An important difference between beef and dairy cattle is the potential period of treatment with antibiotics. Antibiotic supplements are generally used for only limited periods of time in beef cattle, whereas the period of treatment in dairy cattle may extend over many years. General experience has shown that the prolonged use of antibiotics eventually leads to the selection of resistant microbial populations, ultimately negating any positive effects from direct supplementation. Furthermore, concerns in regards to contamination of products with antibiotic residues, antibiotic toxicity, and the spreading of multiple drug resistance are also of concern when administering ionophore antibiotics to animals.
Previously characterised bacteriocins are a heterogeneous group of proteinaceous antibiotics, and are found throughout the family of microorganisms known as bacteria. Subject of particular study are those produced by the lactic acid bacteria and food Propionibacter, organisms with wide use throughout the food processing industry. Bacteriocins,produced by these organisms play a role in the control of Gram Positive bacterial contaminants and food borne pathogens.
The lactic acid bacteria ("LAB"), a diverse group of organisms which includes the Lactobacilli, Lactococci, Pediococci, Leuconostoc, and Streptococci, are of particular interest in terms of the widespread occurrence of bacteriocins within the group. These species are in wide use throughout the fermented dairy, food and meat processing industries. Their role in both the preservation and flavour characteristics of foods has been well documented. Most of the bacteriocins produced by this group are active only against other lactic acid bacteria, but several display anti-bacterial activity towards more phylogenetically distant Gram Positive and, under certain conditions, Gram Negative bacteria. Nisin, a bacteriocin produced by Lactococcus lactis with a very wide spectrum of activity, has found use as an additive in the food processing and animal health industries.
To date, approximately 30 bacteriocins from food related AB have been reported (Klaenhammer, 1993), as well as two more in dairy Propionibacter (Barefoot and Nettles, 1993). In the majority of cases, primary characterisation has been based on the source of isolation, spectrum of activity among LAB and food contaminants, sensitivity to gastric proteases and crude estimates of molecular weight. Only a portion of these have been isolated and well characterised in terms of protein and genetic components.
In a recent review, Klaenhammer (1993) classified the LAB-derived bacteriocins known to date into four major groups:
Class I--Lantibiotics: small peptides (&lt;5 kDa) containing the unusual amino acids lanthionine and .beta.-methyl lanthionine. These are of particular interest in that they have very broad spectra of activity relative to other bacteriocins. Examples include Nisin, Nisin Z, carnocin U 149, lacticin 481 and lactocin 5.
Class II--Small non-lanthionine containing peptides: a heterogeneous group of small peptides (&lt;10 kDa). This group includes peptides active against Lysteria spp.
Class III--Large Heat Labile Proteins: &gt;30 kDa, Helveticin is the only one characterised to date.
Class IV--Complex bacteriocins: proteins containing additional moieties (lipids, carbohydrates). None have yet been purified.
Both Class III and IV generally would be expected to have very narrow spectra of activity.
The primary target of the LAB bacteriocins in susceptible strains appears to be the cell membrane. Effects on target cells include membrane depolarisation, efflux of various ions and cellular constituents, and in some cases cell lysis. All are thought to result from the formation by the bacteriocins of membrane pores. Recent work (Bruno and Montville, 1993) suggests that the primary effect is the dissipation of Proton Motive Force (PMF) in a concentration-dependent fashion. In Gram Positive bacteria, PMF plays a fundamental role in energy metabolism, and dissipation affects both ATP generation and active transport mechanisms, ultimately leading to cell death. This mechanism of activity is similar to that of the ionophore antibiotics.
Secondary effects such as leaking of cell constituents or cellular lysis occur at much higher bacteriocin concentrations. Cell lysis observed with certain bacteriocins like nisin and pediocin PA-1 is a non-specific effect, likely resulting from the induction of cell-associated autolytic systems by displacement of muramidase enzymes associated with the lipoteichoic acid component of the cell wall by these bacteriocins (Piard and Desmazeaud, 1992).
Bacteriocin-producing strains are normally immune to the action of the bacteriocin they produce, indicating the presence of an immunity mechanism. Identification of these immunity proteins by characterisation of bacteriocin producing non-immune mutants has revealed a number of points.
Firstly, immunity proteins are usually cotranscribed with the bacteriocin structural gene.
Secondly, sequence analysis of the open reading frames assigned to immunity indicate that the gene products have the characteristics of membrane-bound proteins. In fact, expression of the nisin immunity protein (nisI), an externally located lipo-protein, was found to confer resistance in a non-producing strain of L. lactis subsp. lactis. (However, recent work on the immunity protein for lactococcin A has shown in this instance the protein may not be membrane bound (Nisen-Meyer et al. 1993)).
Thirdly, evidence from the lactococcin A,B,M producing strain of L. lactis indicates that no cross immunity occurs in deletion mutants missing one of the immunity proteins, indicating that immunity is bacteriocin specific. The functional basis of immune mechanisms has yet to be investigated.
Determinations of spectra of activity of these bacteriocin agents have largely been limited to organisms which occur as contaminants in specific food products, and as of yet no reports concerning sensitivities among rumen bacteria have been published.
To date, limited information concerning bacteriocin production within rumen bacterial isolates is available, and no comprehensive survey of rumen Gram Positive bacteria has been reported. Iverson and Mills (1976) surveyed 47 strains of Streptococcus bovis, of which fourteen were bacteriocin producers. Based on sensitivity profiles, these could be further divided into 6 groups. However, only one of these producers was of rumen origin. Similarly, Arihara et al. (1993) reported a putative bacteriocin in S. bovis ATCC 1388 (which is also not a rumen isolate). No further work beyond protease sensitivity was carried out in either case. However, given the wide occurrence of lantibiotic production among different species of Streptococci, these S. bovis bacteriocins may represent new lantibiotics. A putative bacteriocin has also been identified in a single strain of Ruminococcus albus (Odenyo et al., 1993).
With the exception of the single isolates of Ruminococcus albus (Odenyo et al., 1993) and Streptococcus bovis (Iverson and Mills, 1976), little work has been carried out on the occurrence of bacteriocins among rumen isolates. Given the large numbers and diversity of the bacterial population within the rumen, this represents a valuable environment for the isolation of new bacterlocins. Furthermore, the rumen environment contains a high level of proteolytic activity, which should yield new bacteriocins with interesting protease stabilities.
Butyrivibrio fibrisolvens represents a major component of the Gram Positive rumen flora. However, the designation as a single species is misleading, as the "species" consists of a large number of diverse isolates that show differences in terms of extra-cellular polysaccharides, antigenicity, proteolytic activity and protein composition of the cellular membrane.
In general, speciation of Butyrivibrio fibrisolvens has been based on common features including anaerobiosis, the production of butyric acid as a major metabolic end-product, resistance to Nalidixic acid, and a vibrioid cellular shape (Bryant, 1986). Taxonomic classification of the isolated strains based on comparisons of 16s rRNA sequence, or the DNA sequence of the gene coding for the 16s ribosomal RNA subunit, or SDS gel electrophoresis of soluble proteins is also possible.
Whole cells of Butyrivibrio fibrisolvens stain Gram Negative; however, thin section analysis of the cellular envelope has demonstrated that they are in fact Gram Positive, having an unusually thin peptidoglycan layer. 16s rRNA gene sequence analysis has confirmed that Butyrivibrio spp. are in fact closely related to Gram Positive organisms of the Clostridial cluster XIVa (Forster et al., 1995).
Therefore it is desirable to obtain antibiotics that are proteinaceous but gastric protease resistant, that may exhibit a high degree of specificity with respect to the target microorganisms against which they are effective, that are effective as inhibitors of target microorganisms under anaerobic conditions and ineffective in atmospheric conditions, and that are produced in the rumen of ruminants.
Accordingly, many B. fibrisolvens isolates from both diverse rumen and geographical sources have been examined for the presence of bacteriocins meeting these requirements. A surprisingly high incidence of bacteriocin production has been observed among these isolates.